Heterotandem bicyclic peptide complex

ABSTRACT

The present invention relates to a heterotandem bicyclic peptide complex which comprises a first peptide ligand, which binds to Nectin-4, conjugated via a linker to two second peptide ligands, which bind to CD137. The invention also relates to the use of said heterotandem bicyclic peptide complex in preventing, suppressing or treating cancer.

FIELD OF THE INVENTION

The present invention relates to a heterotandem bicyclic peptide complexwhich comprises a first peptide ligand, which binds to Nectin-4,conjugated via a linker to two second peptide ligands, which bind toCD137. The invention also relates to the use of said heterotandembicyclic peptide complex in preventing, suppressing or treating cancer.

BACKGROUND OF THE INVENTION

Cyclic peptides can bind with high affinity and target specificity toprotein targets and hence are an attractive molecule class for thedevelopment of therapeutics. In fact, several cyclic peptides arealready successfully used in the clinic, as for example theantibacterial peptide vancomycin, the immunosuppressant drugcyclosporine or the anti-cancer drug octreotide (Driggers et al. (2008),Nat Rev Drug Discov 7 (7), 608-24). Good binding properties result froma relatively large interaction surface formed between the peptide andthe target as well as the reduced conformational flexibility of thecyclic structures. Typically, macrocycles bind to surfaces of severalhundred square angstrom, as for example the cyclic peptide CXCR4antagonist CVX15 (400 Å²; Wu et al. (2007), Science 330, 1066-71), acyclic peptide with the Arg-Gly-Asp motif binding to integrin αVb3 (355Å²) (Xiong et al. (2002), Science 296 (5565), 151-5) or the cyclicpeptide inhibitor upain-1 binding to urokinase-type plasminogenactivator (603 Å²; Zhao et al. (2007), J Struct Biol 160 (1), 1-10).

Due to their cyclic configuration, peptide macrocycles are less flexiblethan linear peptides, leading to a smaller loss of entropy upon bindingto targets and resulting in a higher binding affinity. The reducedflexibility also leads to locking target-specific conformations,increasing binding specificity compared to linear peptides. This effecthas been exemplified by a potent and selective inhibitor of matrixmetalloproteinase 8 (MMP-8) which lost its selectivity over other MMPswhen its ring was opened (Cherney et al. (1998), J Med Chem 41 (11),1749-51). The favorable binding properties achieved throughmacrocyclization are even more pronounced in multicyclic peptides havingmore than one peptide ring as for example in vancomycin, nisin andactinomycin.

Different research teams have previously tethered polypeptides withcysteine residues to a synthetic molecular structure (Kemp and McNamara(1985), J. Org. Chem; Timmerman et al. (2005), ChemBioChem). Meloen andco-workers had used tris(bromomethyl)benzene and related molecules forrapid and quantitative cyclisation of multiple peptide loops ontosynthetic scaffolds for structural mimicry of protein surfaces(Timmerman et al. (2005), ChemBioChem).

Methods for the generation of candidate drug compounds wherein saidcompounds are generated by linking cysteine containing polypeptides to amolecular scaffold as for example1,1′,1″-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA) aredisclosed in WO 2019/122860 and WO 2019/122863.

Phage display-based combinatorial approaches have been developed togenerate and screen large libraries of bicyclic peptides to targets ofinterest (Heinis et al. (2009), Nat Chem Biol 5 (7), 502-7 and WO2009/098450). Briefly, combinatorial libraries of linear peptidescontaining three cysteine residues and two regions of six random aminoacids (Cys-(Xaa)₆-Cys-(Xaa)₆-Cys) were displayed on phage and cyclisedby covalently linking the cysteine side chains to a small molecule(tris-(bromomethyl)benzene).

SUMMARY OF THE INVENTION

According to a first aspect of the invention, there is provided aheterotandem bicyclic peptide complex comprising:

-   -   (a) a first peptide ligand which binds to Nectin-4 and which has        the sequence C_(i)P[1Nal][dD]C_(ii)M[HArg]DWSTP[HyP]WC_(ii) (SEQ        ID NO: 1; BCY8116); conjugated via an        N-(acid-PEG₃)-N-bis(PEG₃-azide) linker to    -   (b) two second peptide ligands which bind to CD137 both of which        have the sequence        Ac—C_(i)[tBuAla]PE[D-Lys(PYA)]PYC_(ii)FADPY[Nle]C_(iii)-A (SEQ        ID NO: 2; BCY8928); wherein each of said peptide ligands        comprise a polypeptide comprising three reactive cysteine groups        (C_(i), C_(ii) and C_(iii)), separated by two loop sequences,        and a molecular scaffold which is        1,1′,1″-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA)        and which forms covalent bonds with the reactive cysteine groups        of the polypeptide such that two polypeptide loops are formed on        the molecular scaffold;        wherein Ac represents acetyl, HArg represents homoarginine, HyP        represents trans-4-hydroxy-L-proline, 1Nal represents        1-naphthylalanine, tBuAla represents t-butyl-alanine, PYA        represents 4-pentynoic acid and Nle represents norleucine.

According to a further aspect of the invention, there is provided apharmaceutical composition comprising a heterotandem bicyclic peptidecomplex as defined herein in combination with one or morepharmaceutically acceptable excipients.

According to a further aspect of the invention, there is provided aheterotandem bicyclic peptide complex as defined herein for use inpreventing, suppressing or treating cancer.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: (A) Analysis of the Nectin-4/CD137 heterotandem bicyclic peptidecomplex in the Promega CD137 luciferase reporter assay in the presenceof Nectin-4 expressing H292 cells. BCY11617 is a heterotandem bicyclicpeptide complex that binds to Nectin-4 with the same affinity asBCY11863 but that does not bind to CD137. (B) Summary of EC50 (nM) ofBCY11863 in the Promega CD137 luciferase reporter assay in coculturewith different cell lines that express Nectin-4 endogenously or areengineered to overexpress Nectin-4.

FIG. 2: Nectin-4/CD137 heterotandem bicyclic peptide complexes induceIFN-γ (FIG. 2A) and IL-2 (FIG. 2B) cytokine secretion in a PBMC-4T1co-culture assay. 4T1 cells were engineered to express Nectin-4.BCY11617 is a heterotandem bicyclic peptide complex that binds toNectin-4 with the same affinity as BCY11863 but does not bind to CD137.FIG. 2C represents a summary of EC50 (nM) of BCY11863 in the cytokinesecretion assay with multiple human PBMC donors and tumor cell lines.

FIG. 3: Pharmacokinetics of heterotandem bicyclic peptide complexBCY11863 in SD Rats and Cynomolgus monkey (cyno) dosed IV at 2 mg/kg(n=3) and 1 mg/kg (n=2) respectively.

FIG. 4: Anti-tumor activity of BCY11863 in a syngeneic mouse Nectin-4overexpressing MC38 tumor model (MC38#13). Tumor volumes during andafter BCY11863 treatment. Number of complete responder (CR) mice on D69are indicated in parentheses. QD: daily dosing; Q3D: every three daysdosing; ip: intraperitoneal administration.

FIG. 5: BCY11863 treatment leads to an immunogenic memory to Nectin-4overexpressing MC38 tumor cells (MC38#13). Tumor volumes are shown afterinoculation to naïve C57BL/6J-hCD137 mice or mice that had completeresponses (CR) to BCY11863. Note that none of the CR mice developedtumors by the end of the observation period (22 days).

FIG. 6: BCY11863 demonstrates anti-tumor activity in a mouse syngeneicNectin-4 overexpressing CT26 tumor model (CT26#7). Tumor volumes duringBCY11863 treatment. Q3D: every three days dosing; ip: intraperitonealadministration.

FIG. 7: Total T cells and CD8+ T cells increase in CT26#7 tumor tissue1h after the last (6th) Q3D dose of BCY11863. Analysis of (A) total Tcells, CD8+ T cells, CD4+ T cells, Tregs and (B) CD8+ T cell/Treg-ratioin CT26#7 tumor tissue 1h after last Q3D dose of BCY11863.

FIG. 8: Pharmacokinetic profiles of BCY11863 in plasma and tumor tissueof CT26#7 syngeneic tumor bearing animals after a single intravenous(iv) administration of 5 mg/kg of BCY11863.

FIG. 9: Plasma concentration vs time curve of BCY11863 from a 15 mg/kgintraperitoneal dose in CD-1 mice (n=3) and the terminal plasma halflife for BCY11863.

FIG. 10: Surface plasmon resonance (SPR) binding study of BCY11863 toimmobilized (A) Nectin-4 and (B) CD137. Dual binding SPR assayimmobilizing (C) CD137 and (D) Nectin-4 on the SPR chip followed bycapturing BCY11863. The affinity of bound BCY11863 to soluble humanNectin-4 (C) or CD137 (D) is measured by flowing the soluble receptorover the chip at different concentrations. (E) Binding of BCY13582(biotinylated BCY11863) immobilized on streptavidin SPR chip to solublehuman CD137.

FIG. 11: Retrogenix's cell microarray technology used to explorenon-specific off target interactions of BCY13582 (biotinylatedBCY11863). Shown here is screening data that shows that 1 μM of BCY13582added to microarray slides expressing 11 different proteins only bindsto CD137 and Nectin-4 (detected using AlexaFluor647 labelledstreptavidin). The binding signal is displaced when incubated withBCY11863.

FIG. 12: Tumor growth curves of MC38#13 tumors in huCD137 C57Bl/6 micedemonstrate the anti-tumor activity of BCY11863 after different dosesand dose intervals. The number of complete responder animals (CR; nopalpable tumor) on day 15 after treatment initiation is indicated inparentheses.

FIG. 13: Tumor growth curves (mean±SEM) of MC38#13 tumors (n=6/cohort)in huCD137 C57Bl/6 mice demonstrate the anti-tumor activity of BCY11863at different doses and dose schedules. The number of complete responderanimals (CR; no palpable tumor) on day 52 after treatment initiation isindicated in parentheses. (A) Cohorts dosed with vehicle or 3 mg/kgtotal weekly dose of BCY11863. (B) Cohorts dosed with vehicle or 10mg/kg total weekly dose of BCY11863. (C) Cohorts dosed with vehicle or30 mg/kg total weekly dose of BCY11863.

FIG. 14: Pharmacokinetics of heterotandem bicyclic peptide complexBCY11863 in SD Rats dosed IV at 100 mg/kg (n=3) and measurement ofconcentration of BCY11863 and potential metabolites BCY15155 andBCY14602 in plasma.

DETAILED DESCRIPTION OF THE INVENTION

According to a first aspect of the invention, there is provided aheterotandem bicyclic peptide complex comprising:

-   -   (a) a first peptide ligand which binds to Nectin-4 and which has        the sequence C_(i)P[1Nal][dD]C_(ii)M[HArg]DWSTP[HyP]WC_(iii)        (SEQ ID NO: 1; BCY8116); conjugated via an        N-(acid-PEG₃)-N-bis(PEG₃-azide) linker to    -   (b) two second peptide ligands which bind to CD137 both of which        have the sequence        Ac—C_(i)[tBuAla]PE[D-Lys(PYA)]PYC_(ii)FADPY[Nle]C_(iii)-A (SEQ        ID NO: 2; BCY8928); wherein each of said peptide ligands        comprise a polypeptide comprising three reactive cysteine groups        (C_(i), C_(ii) and C_(iii)), separated by two loop sequences,        and a molecular scaffold which is        1,1′,1″-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA)        and which forms covalent bonds with the reactive cysteine groups        of the polypeptide such that two polypeptide loops are formed on        the molecular scaffold;        wherein Ac represents acetyl, HArg represents homoarginine, HyP        represents trans-4-hydroxy-L-proline, 1Nal represents        1-naphthylalanine, tBuAla represents t-butyl-alanine, PYA        represents 4-pentynoic acid and Nle represents norleucine.

References herein to a N-(acid-PEG₃)-N-bis(PEG₃-azide) linker include:

In one embodiment, the heterotandem bicyclic peptide complex isBCY11863:

Full details of BCY11863 are shown in Table A below:

TABLE A Composition of BCY11863 Complex Nectin-4 Attachment CD137Attachment No. BCY No. Point Linker BCY Nos. Point BCY11863 BCY8116N-terminus N-(acid- BCY8928, dLys (PYA)4 PEG3)- BCY8928 N-bis (PEG3-azide)

Data is presented herein in FIG. 1 and Table 1 which shows that BCY11863demonstrated strong CD137 activation in a CD137 reporter assay. Inaddition, data is presented herein in FIG. 2 and Table 2 which showsthat BCY11863 induces robust IL-2 and IFN-γ cytokine secretion in a PBMCco-culture assays with multiple tumor cell lines and human PBMC donors.Furthermore, data is presented herein in FIG. 3 and Table 5 which showsthat BCY11863 demonstrated an excellent PK profile with a terminalhalf-life of 4.1 hours in SD Rats and 5.3 hours in cyno. Data shown inFIGS. 10 and 11 along with methods section 11 and 12 demonstrate bindingand exquisite selectivity of BCY11863 for its target Nectin-4 and CD137.FIGS. 4 and 5 demonstrate profound anti-tumor activity of BCY11863 inMC38#13 syngeneic mice and the formation of immunogenic memory afterBCY11863 treatment. FIGS. 6 and 7 demonstrate anti-tumor activity ofBCY11863 in CT26#7 syngeneic model with corresponding infiltration ofcytotoxic T cells into the tumor. FIGS. 12 and 13 clearly demonstratethat BCY11863 does not have to maintain measurable plasma concentrationsas dosing with 1.5 mg/kg BIW and 5 mg/kg at 0, 24 h in a week producedrobust anti-tumor activity.

Reference herein is made to certain analogues (i.e. modifiedderivatives) and metabolites of BCY11863, each of which form additionalaspects of the invention and are summarised in Table B below:

TABLE B Composition of BCY11863 analoques and metabolites ComplexNectin-4 Attachment CD137 Attachment No. BCY No. Point Linker BCY No.Point Modifier BCY13390 BCY8116 N-terminus N-(acid-PEG₃)-N- BCY8928,dLys(PYA)4 bis(PEG₃-azide) BCY13389 dLys(PYA)4 BCY13582 BCY8116N-terminus N-(acid-PEG₃)-N- BCY8928, dLys(PYA)4 Biotin- bis(PEG₃-azide)BCY13389 dLys(PYA)4 Peg 12 BCY13583 BCY8116 N-terminus N-(acid-PEG₃)-N-BCY8928, dLys(PYA)4 Alexa bis(PEG₃-azide) BCY13389 dLys(PYA)4 Fluor 488BCY13628 BCY8116 N-terminus N-(acid-PEG₃)-N- BCY8928, dLys(PYA)4 Cyanine5 bis(PEG₃-azide) BCY13389 dLys(PYA)4 BCY15155 BCY8116 N-terminusN-(acid-PEG₃)-N- BCY8928, dLys(PYA)4 bis(PEG₃-azide) BCY14601 dLys(PYA)4BCY14602 BCY8116 N-terminus N-(acid-PEG₃)-N- BCY14601 dLys(PYA)4bis(PEG₃-azide)wherein BCY14601 represents a bicyclic peptide ligand having thesequence of C_(i)[tBuAla]PE[D-Lys(PYA)]PYC_(ii)FADPY[Nle]C_(iii)-A (SEQID NO: 3) with TATA as a molecular scaffold;and wherein BCY13389 represents a bicyclic peptide ligand having thesequence of [Ac]C_(i)[tBuAla]PE[D-Lys(PYA)]PYC_(ii)FADPY[Nle]C_(iii)—K(SEQ ID NO: 4) with TATA as a molecular scaffold.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art, such as in the arts of peptide chemistry, cell culture andphage display, nucleic acid chemistry and biochemistry. Standardtechniques are used for molecular biology, genetic and biochemicalmethods (see Sambrook et al., Molecular Cloning: A Laboratory Manual,3rd ed., 2001, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y.; Ausubel et al., Short Protocols in Molecular Biology (1999) 4^(th)ed., John Wiley & Sons, Inc.), which are incorporated herein byreference.

Nomenclature Numbering

When referring to amino acid residue positions within compounds of theinvention, cysteine residues (C_(i), C_(ii) and C_(iii)) are omittedfrom the numbering as they are invariant, therefore, the numbering ofamino acid residues within SEQ ID NO: 1 is referred to as below:

(SEQ ID NO: 1) C_(i)-P₁-1Nal₂-dD₃-C_(ii)-M₄-HArg₅-D₆-W₇-S₈-T₉-P₁₀-HyP₁₁-W₁₂-C_(iii).

For the purpose of this description, the bicyclic peptides are cyclisedwith 1,1′,1″-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA) andyielding a tri-substituted structure. Cyclisation with TATA occurs onC_(i), C_(ii), and C_(iii).

Molecular Format

N- or C-terminal extensions to the bicycle core sequence are added tothe left or right side of the sequence, separated by a hyphen. Forexample, an N-terminal βAla-Sar10-Ala tail would be denoted as:

βAla-Sar10-A-(SEQ ID NO: X).

Inversed Peptide Sequences

In light of the disclosure in Nair et al (2003) J Immunol 170(3),1362-1373, it is envisaged that the peptide sequences disclosed hereinwould also find utility in their retro-inverso form. For example, thesequence is reversed (i.e. N-terminus becomes C-terminus and vice versa)and their stereochemistry is likewise also reversed (i.e. D-amino acidsbecome L-amino acids and vice versa). For the avoidance of doubt,references to amino acids either as their full name or as their aminoacid single or three letter codes are intended to be represented hereinas L-amino acids unless otherwise stated. If such an amino acid isintended to be represented as a D-amino acid then the amino acid will beprefaced with a lower case d within square parentheses, for example[dA], [dD], [dE], [dK], [d1Nal], [dNle], etc.

Advantages of the Peptide Ligands

Certain heterotandem bicyclic peptide complexes of the present inventionhave a number of advantageous properties which enable them to beconsidered as suitable drug-like molecules for injection, inhalation,nasal, ocular, oral or topical administration. Such advantageousproperties include:

-   -   Species cross-reactivity. This is a typical requirement for        preclinical pharmacodynamics and pharmacokinetic evaluation;    -   Protease stability. Heterotandem bicyclic peptide complexes        should ideally demonstrate stability to plasma proteases,        epithelial (“membrane-anchored”) proteases, gastric and        intestinal proteases, lung surface proteases, intracellular        proteases and the like. Protease stability should be maintained        between different species such that a heterotandem bicyclic        peptide lead candidate can be developed in animal models as well        as administered with confidence to humans;    -   Desirable solubility profile. This is a function of the        proportion of charged and hydrophilic versus hydrophobic        residues and intra/inter-molecular H-bonding, which is important        for formulation and absorption purposes;    -   Selectivity. Certain heterotandem bicyclic peptide complexes of        the invention demonstrate good selectivity over other targets;    -   An optimal plasma half-life in the circulation. Depending upon        the clinical indication and treatment regimen, it may be        required to develop a heterotandem bicyclic peptide complex for        short exposure in an acute illness management setting, or        develop a heterotandem bicyclic peptide complex with enhanced        retention in the circulation, and is therefore optimal for the        management of more chronic disease states. Other factors driving        the desirable plasma half-life are requirements of sustained        exposure for maximal therapeutic efficiency versus the        accompanying toxicology due to sustained exposure of the agent.

Crucially, data is presented herein where the heterotandem bicyclicpeptide complex of the invention demonstrates anti-tumor efficacy whendosed at a frequency that does not maintain plasma concentrations abovethe in vitro EC₅₀ of the compound. This is in contrast to largerrecombinant biologic (i.e. antibody based) approaches to CD137 agonismor bispecific CD137 agonism (Segal et al., Clin Cancer Res.,23(8):1929-1936 (2017), Claus et al., Sci Trans Med., 11 (496):eaav5989, 1-12 (2019), Hinner et al., Clin Cancer Res., 25(19):5878-5889(2019)). Without being bound by theory, the reason for this observationis thought to be due to the fact that heterotandem bicycle complexeshave relatively low molecular weight (typically <15 kDa), they are fullysynthetic and they are tumor targeted agonists of CD137. As such, theyhave relatively short plasma half lives but good tumor penetrance andretention. Data is presented herein which fully supports theseadvantages. For example, anti-tumor efficacy in syngeneic rodent modelsin mice with humanized CD137 is demonstrated either daily or every3^(rd) day. In addition, intraperitoneal pharmacokinetic data shows thatthe plasma half life is <3 hours, which would predict that thecirculating concentration of the complex would consistently drop belowthe in vitro EC₅₀ between doses. Furthermore, tumor pharmacokinetic datashows that levels of heterotandem bicycle complex in tumor tissue may behigher and more sustained as compared to plasma levels.

It will be appreciated that this observation forms an important furtheraspect of the invention. Thus, according to a further aspect of theinvention, there is provided a method of treating cancer which comprisesadministration of a heterotandem bicyclic peptide complex as definedherein at a dosage frequency which does not sustain plasmaconcentrations of said complex above the in vitro EC₅₀ of said complex.

-   -   Immune Memory. Coupling the cancer cell binding bicyclic peptide        ligand with the immune cell binding bicyclic peptide ligand        provides the synergistic advantage of immune memory. Data is        presented herein which demonstrates that the heterotandem        bicyclic peptide complex of the invention not only eradicates        tumors but upon readministration of the tumorigenic agent, none        of the inoculated complete responder mice developed tumors (see        FIG. 5). This indicates that treatment with the selected        heterotandem bicyclic peptide complex of the invention has        induced immunogenic memory in the complete responder mice. This        has a significant clinical advantage in order to prevent        recurrence of said tumor once it has been initially controlled        and eradicated.

Peptide Ligands

A peptide ligand, as referred to herein, refers to a peptide covalentlybound to a molecular scaffold. Typically, such peptides comprise two ormore reactive groups (i.e. cysteine residues) which are capable offorming covalent bonds to the scaffold, and a sequence subtended betweensaid reactive groups which is referred to as the loop sequence, since itforms a loop when the peptide is bound to the scaffold. In the presentcase, the peptides comprise at least three reactive groups selected fromcysteine, 3-mercaptopropionic acid and/or cysteamine and form at leasttwo loops on the scaffold.

Pharmaceutically Acceptable Salts

It will be appreciated that salt forms are within the scope of thisinvention, and references to peptide ligands include the salt forms ofsaid ligands.

The salts of the present invention can be synthesized from the parentcompound that contains a basic or acidic moiety by conventional chemicalmethods such as methods described in Pharmaceutical Salts: Properties,Selection, and Use, P. Heinrich Stahl (Editor), Camille G. Wermuth(Editor), ISBN: 3-90639-026-8, Hardcover, 388 pages, August 2002.Generally, such salts can be prepared by reacting the free acid or baseforms of these compounds with the appropriate base or acid in water orin an organic solvent, or in a mixture of the two.

Acid addition salts (mono- or di-salts) may be formed with a widevariety of acids, both inorganic and organic. Examples of acid additionsalts include mono- or di-salts formed with an acid selected from thegroup consisting of acetic, 2,2-dichloroacetic, adipic, alginic,ascorbic (e.g. L-ascorbic), L-aspartic, benzenesulfonic, benzoic,4-acetamidobenzoic, butanoic, (+) camphoric, camphor-sulfonic,(+)-(1S)-camphor-10-sulfonic, capric, caproic, caprylic, cinnamic,citric, cyclamic, dodecylsulfuric, ethane-1,2-disulfonic,ethanesulfonic, 2-hydroxyethanesulfonic, formic, fumaric, galactaric,gentisic, glucoheptonic, D-gluconic, glucuronic (e.g. D-glucuronic),glutamic (e.g. L-glutamic), α-oxoglutaric, glycolic, hippuric,hydrohalic acids (e.g. hydrobromic, hydrochloric, hydriodic),isethionic, lactic (e.g. (+)-L-lactic, (±)-DL-lactic), lactobionic,maleic, malic, (−)-L-malic, malonic, (±)-DL-mandelic, methanesulfonic,naphthalene-2-sulfonic, naphthalene-1,5-disulfonic,1-hydroxy-2-naphthoic, nicotinic, nitric, oleic, orotic, oxalic,palmitic, pamoic, phosphoric, propionic, pyruvic, L-pyroglutamic,salicylic, 4-amino-salicylic, sebacic, stearic, succinic, sulfuric,tannic, (+)-L-tartaric, thiocyanic, p-toluenesulfonic, undecylenic andvaleric acids, as well as acylated amino acids and cation exchangeresins.

One particular group of salts consists of salts formed from acetic,hydrochloric, hydriodic, phosphoric, nitric, sulfuric, citric, lactic,succinic, maleic, malic, isethionic, fumaric, benzenesulfonic,toluenesulfonic, sulfuric, methanesulfonic (mesylate), ethanesulfonic,naphthalenesulfonic, valeric, propanoic, butanoic, malonic, glucuronicand lactobionic acids. One particular salt is the hydrochloride salt.Another particular salt is the acetate salt.

If the compound is anionic, or has a functional group which may beanionic (e.g., —COOH may be —COO⁻), then a salt may be formed with anorganic or inorganic base, generating a suitable cation. Examples ofsuitable inorganic cations include, but are not limited to, alkali metalions such as Li⁺, Na⁺ and K⁺, alkaline earth metal cations such as Ca²⁺and Mg²⁺, and other cations such as Al³⁺ or Zn⁺. Examples of suitableorganic cations include, but are not limited to, ammonium ion (i.e., NH₄⁺) and substituted ammonium ions (e.g., NH₃R⁺, NH₂R₂ ⁺, NHR₃ ⁺, NR₄ ⁺).Examples of some suitable substituted ammonium ions are those derivedfrom: methylamine, ethylamine, diethylamine, propylamine,dicyclohexylamine, triethylamine, butylamine, ethylenediamine,ethanolamine, diethanolamine, piperazine, benzylamine,phenylbenzylamine, choline, meglumine, and tromethamine, as well asamino acids, such as lysine and arginine. An example of a commonquaternary ammonium ion is N(CH₃)₄ ⁺.

Where the compounds of the invention contain an amine function, thesemay form quaternary ammonium salts, for example by reaction with analkylating agent according to methods well known to the skilled person.Such quaternary ammonium compounds are within the scope of theinvention.

Modified Derivatives

It will be appreciated that modified derivatives of the peptide ligandsas defined herein are within the scope of the present invention.Examples of such suitable modified derivatives include one or moremodifications selected from: N-terminal and/or C-terminal modifications;replacement of one or more amino acid residues with one or morenon-natural amino acid residues (such as replacement of one or morepolar amino acid residues with one or more isosteric or isoelectronicamino acids; replacement of one or more non-polar amino acid residueswith other non-natural isosteric or isoelectronic amino acids); additionof a spacer group; replacement of one or more oxidation sensitive aminoacid residues with one or more oxidation resistant amino acid residues;replacement of one or more amino acid residues with an alanine,replacement of one or more L-amino acid residues with one or moreD-amino acid residues; N-alkylation of one or more amide bonds withinthe bicyclic peptide ligand; replacement of one or more peptide bondswith a surrogate bond; peptide backbone length modification;substitution of the hydrogen on the alpha-carbon of one or more aminoacid residues with another chemical group, modification of amino acidssuch as cysteine, lysine, glutamate/aspartate and tyrosine with suitableamine, thiol, carboxylic acid and phenol-reactive reagents so as tofunctionalise said amino acids, and introduction or replacement of aminoacids that introduce orthogonal reactivities that are suitable forfunctionalisation, for example azide or alkyne-group bearing amino acidsthat allow functionalisation with alkyne or azide-bearing moieties,respectively.

In one embodiment, the modified derivative comprises an N-terminaland/or C-terminal modification. In a further embodiment, wherein themodified derivative comprises an N-terminal modification using suitableamino-reactive chemistry, and/or C-terminal modification using suitablecarboxy-reactive chemistry. In a further embodiment, said N-terminal orC-terminal modification comprises addition of an effector group,including but not limited to a cytotoxic agent, a radiochelator or achromophore.

In a further embodiment, the modified derivative comprises an N-terminalmodification. In a further embodiment, the N-terminal modificationcomprises an N-terminal acetyl group. In this embodiment, the N-terminalcysteine group (the group referred to herein as C_(i)) is capped withacetic anhydride or other appropriate reagents during peptide synthesisleading to a molecule which is N-terminally acetylated. This embodimentprovides the advantage of removing a potential recognition point foraminopeptidases and avoids the potential for degradation of the bicyclicpeptide.

In an alternative embodiment, the N-terminal modification comprises theaddition of a molecular spacer group which facilitates the conjugationof effector groups and retention of potency of the bicyclic peptide toits target.

In a further embodiment, the modified derivative comprises a C-terminalmodification. In a further embodiment, the C-terminal modificationcomprises an amide group. In this embodiment, the C-terminal cysteinegroup (the group referred to herein as C_(ii)) is synthesized as anamide during peptide synthesis leading to a molecule which isC-terminally amidated. This embodiment provides the advantage ofremoving a potential recognition point for carboxypeptidase and reducesthe potential for proteolytic degradation of the bicyclic peptide.

In one embodiment, the modified derivative comprises replacement of oneor more amino acid residues with one or more non-natural amino acidresidues. In this embodiment, non-natural amino acids may be selectedhaving isosteric/isoelectronic side chains which are neither recognisedby degradative proteases nor have any adverse effect upon targetpotency.

Alternatively, non-natural amino acids may be used having constrainedamino acid side chains, such that proteolytic hydrolysis of the nearbypeptide bond is conformationally and sterically impeded. In particular,these concern proline analogues, bulky sidechains, Ca-disubstitutedderivatives (for example, aminoisobutyric acid, Aib), and cyclo aminoacids, a simple derivative being amino-cyclopropylcarboxylic acid.

In one embodiment, the modified derivative comprises the addition of aspacer group. In a further embodiment, the modified derivative comprisesthe addition of a spacer group to the N-terminal cysteine (C_(i)) and/orthe C-terminal cysteine (C_(iii)).

In one embodiment, the modified derivative comprises replacement of oneor more oxidation sensitive amino acid residues with one or moreoxidation resistant amino acid residues. In a further embodiment, themodified derivative comprises replacement of a tryptophan residue with anaphthylalanine or alanine residue. This embodiment provides theadvantage of improving the pharmaceutical stability profile of theresultant bicyclic peptide ligand.

In one embodiment, the modified derivative comprises replacement of oneor more charged amino acid residues with one or more hydrophobic aminoacid residues. In an alternative embodiment, the modified derivativecomprises replacement of one or more hydrophobic amino acid residueswith one or more charged amino acid residues. The correct balance ofcharged versus hydrophobic amino acid residues is an importantcharacteristic of the bicyclic peptide ligands. For example, hydrophobicamino acid residues influence the degree of plasma protein binding andthus the concentration of the free available fraction in plasma, whilecharged amino acid residues (in particular arginine) may influence theinteraction of the peptide with the phospholipid membranes on cellsurfaces. The two in combination may influence half-life, volume ofdistribution and exposure of the peptide drug, and can be tailoredaccording to the clinical endpoint. In addition, the correct combinationand number of charged versus hydrophobic amino acid residues may reduceirritation at the injection site (if the peptide drug has beenadministered subcutaneously).

In one embodiment, the modified derivative comprises replacement of oneor more L-amino acid residues with one or more D-amino acid residues.This embodiment is believed to increase proteolytic stability by sterichindrance and by a propensity of D-amino acids to stabilise 3-turnconformations (Tugyi et al (2005) PNAS, 102(2), 413-418).

In one embodiment, the modified derivative comprises removal of anyamino acid residues and substitution with alanines. This embodimentprovides the advantage of removing potential proteolytic attack site(s).

It should be noted that each of the above mentioned modifications serveto deliberately improve the potency or stability of the peptide. Furtherpotency improvements based on modifications may be achieved through thefollowing mechanisms:

-   -   Incorporating hydrophobic moieties that exploit the hydrophobic        effect and lead to lower off rates, such that higher affinities        are achieved;    -   Incorporating charged groups that exploit long-range ionic        interactions, leading to faster on rates and to higher        affinities (see for example Schreiber et al, Rapid,        electrostatically assisted association of proteins (1996),        Nature Struct. Biol. 3, 427-31); and    -   Incorporating additional constraint into the peptide, by for        example constraining side chains of amino acids correctly such        that loss in entropy is minimal upon target binding,        constraining the torsional angles of the backbone such that loss        in entropy is minimal upon target binding and introducing        additional cyclisations in the molecule for identical reasons.        (for reviews see Gentilucci et al, Curr. Pharmaceutical Design,        (2010), 16, 3185-203, and Nestor et al, Curr. Medicinal Chem        (2009), 16, 4399-418).

Isotopic Variations

The present invention includes all pharmaceutically acceptable(radio)isotope-labeled peptide ligands of the invention, wherein one ormore atoms are replaced by atoms having the same atomic number, but anatomic mass or mass number different from the atomic mass or mass numberusually found in nature, and peptide ligands of the invention, whereinmetal chelating groups are attached (termed “effector”) that are capableof holding relevant (radio)isotopes, and peptide ligands of theinvention, wherein certain functional groups are covalently replacedwith relevant (radio)isotopes or isotopically labelled functionalgroups.

Examples of isotopes suitable for inclusion in the peptide ligands ofthe invention comprise isotopes of hydrogen, such as ²H (D) and ³H (T),carbon, such as ¹¹C, ¹³C and ¹⁴C, chlorine, such as ³⁶Cl, fluorine, suchas ¹⁸F, iodine, such as ¹²³I, ¹²¹I and ¹³¹I, nitrogen, such as ¹³N and¹⁵N, oxygen, such as ¹⁵O, ¹⁷O and ¹⁸O, phosphorus, such as ³²P, sulfur,such as ³⁵S, copper, such as ⁶⁴Cu, gallium, such as ⁶⁷Ga or ⁶⁸Ga,yttrium, such as ⁹⁰Y and lutetium, such as ¹⁷⁷Lu, and Bismuth, such as²¹³Bi.

Certain isotopically-labelled peptide ligands of the invention, forexample, those incorporating a radioactive isotope, are useful in drugand/or substrate tissue distribution studies, and to clinically assessthe presence and/or absence of the Nectin-4 target on diseased tissues.The peptide ligands of the invention can further have valuablediagnostic properties in that they can be used for detecting oridentifying the formation of a complex between a labelled compound andother molecules, peptides, proteins, enzymes or receptors. The detectingor identifying methods can use compounds that are labelled withlabelling agents such as radioisotopes, enzymes, fluorescent substances,luminous substances (for example, luminol, luminol derivatives,luciferin, aequorin and luciferase), etc. The radioactive isotopestritium, i.e. ³H (T), and carbon-14, i.e. ¹⁴C, are particularly usefulfor this purpose in view of their ease of incorporation and ready meansof detection.

Substitution with heavier isotopes such as deuterium, i.e. ²H (D), mayafford certain therapeutic advantages resulting from greater metabolicstability, for example, increased in vivo half-life or reduced dosagerequirements, and hence may be preferred in some circumstances.

Substitution with positron emitting isotopes, such as ¹¹C, ¹⁸F, ¹⁵O and¹³N, can be useful in Positron Emission Topography (PET) studies forexamining target occupancy.

Isotopically-labeled compounds of peptide ligands of the invention cangenerally be prepared by conventional techniques known to those skilledin the art or by processes analogous to those described in theaccompanying Examples using an appropriate isotopically-labeled reagentin place of the non-labeled reagent previously employed.

Synthesis

The peptides of the present invention may be manufactured syntheticallyby standard techniques followed by reaction with a molecular scaffold invitro. When this is performed, standard chemistry may be used. Thisenables the rapid large scale preparation of soluble material forfurther downstream experiments or validation. Such methods could beaccomplished using conventional chemistry such as that disclosed inTimmerman et al (supra).

Thus, the invention also relates to manufacture of polypeptides orconjugates selected as set out herein, wherein the manufacture comprisesoptional further steps as explained below. In one embodiment, thesesteps are carried out on the end product polypeptide/conjugate made bychemical synthesis.

Optionally amino acid residues in the polypeptide of interest may besubstituted when manufacturing a conjugate or complex.

Peptides can also be extended, to incorporate for example another loopand therefore introduce multiple specificities.

To extend the peptide, it may simply be extended chemically at itsN-terminus or C-terminus or within the loops using orthogonallyprotected lysines (and analogues) using standard solid phase or solutionphase chemistry. Standard (bio)conjugation techniques may be used tointroduce an activated or activatable N- or C-terminus. Alternativelyadditions may be made by fragment condensation or native chemicalligation e.g. as described in (Dawson et al. 1994. Synthesis of Proteinsby Native Chemical Ligation. Science 266:776-779), or by enzymes, forexample using subtiligase as described in (Chang et al. Proc Natl AcadSci USA. 1994 Dec. 20; 91(26):12544-8 or in Hikari et al Bioorganic &Medicinal Chemistry Letters Volume 18, Issue 22, 15 Nov. 2008, Pages6000-6003).

Alternatively, the peptides may be extended or modified by furtherconjugation through disulphide bonds. This has the additional advantageof allowing the first and second peptides to dissociate from each otheronce within the reducing environment of the cell. In this case, themolecular scaffold (e.g. TATA) could be added during the chemicalsynthesis of the first peptide so as to react with the three cysteinegroups; a further cysteine or thiol could then be appended to the N orC-terminus of the first peptide, so that this cysteine or thiol onlyreacted with a free cysteine or thiol of the second peptides, forming adisulfide-linked bicyclic peptide-peptide conjugate.

Similar techniques apply equally to the synthesis/coupling of twobicyclic and bispecific macrocycles, potentially creating atetraspecific molecule.

Furthermore, addition of other functional groups or effector groups maybe accomplished in the same manner, using appropriate chemistry,coupling at the N- or C-termini or via side chains. In one embodiment,the coupling is conducted in such a manner that it does not block theactivity of either entity.

Pharmaceutical Compositions

According to a further aspect of the invention, there is provided apharmaceutical composition comprising a peptide ligand as defined hereinin combination with one or more pharmaceutically acceptable excipients.

Generally, the present peptide ligands will be utilised in purified formtogether with pharmacologically appropriate excipients or carriers.Typically, these excipients or carriers include aqueous oralcoholic/aqueous solutions, emulsions or suspensions, including salineand/or buffered media. Parenteral vehicles include sodium chloridesolution, Ringer's dextrose, dextrose and sodium chloride and lactatedRinger's. Suitable physiologically-acceptable adjuvants, if necessary tokeep a polypeptide complex in suspension, may be chosen from thickenerssuch as carboxymethylcellulose, polyvinylpyrrolidone, gelatin andalginates.

Intravenous vehicles include fluid and nutrient replenishers andelectrolyte replenishers, such as those based on Ringer's dextrose.Preservatives and other additives, such as antimicrobials, antioxidants,chelating agents and inert gases, may also be present (Mack (1982)Remington's Pharmaceutical Sciences, 16th Edition).

The peptide ligands of the present invention may be used as separatelyadministered compositions or in conjunction with other agents. These caninclude antibodies, antibody fragments and various immunotherapeuticdrugs, such as cylcosporine, methotrexate, adriamycin or cisplatinum andimmunotoxins. Pharmaceutical compositions can include “cocktails” ofvarious cytotoxic or other agents in conjunction with the proteinligands of the present invention, or even combinations of selectedpolypeptides according to the present invention having differentspecificities, such as polypeptides selected using different targetligands, whether or not they are pooled prior to administration.

The route of administration of pharmaceutical compositions according tothe invention may be any of those commonly known to those of ordinaryskill in the art. For therapy, the peptide ligands of the invention canbe administered to any patient in accordance with standard techniques.The administration can be by any appropriate mode, includingparenterally, intravenously, intramuscularly, intraperitoneally,transdermally, via the pulmonary route, or also, appropriately, bydirect infusion with a catheter. Preferably, the pharmaceuticalcompositions according to the invention will be administered byinhalation. The dosage and frequency of administration will depend onthe age, sex and condition of the patient, concurrent administration ofother drugs, counterindications and other parameters to be taken intoaccount by the clinician.

The peptide ligands of this invention can be lyophilised for storage andreconstituted in a suitable carrier prior to use. This technique hasbeen shown to be effective and art-known lyophilisation andreconstitution techniques can be employed. It will be appreciated bythose skilled in the art that lyophilisation and reconstitution can leadto varying degrees of activity loss and that levels may have to beadjusted upward to compensate.

The compositions containing the present peptide ligands or a cocktailthereof can be administered for prophylactic and/or therapeutictreatments. In certain therapeutic applications, an adequate amount toaccomplish at least partial inhibition, suppression, modulation,killing, or some other measurable parameter, of a population of selectedcells is defined as a “therapeutically-effective dose”. Amounts neededto achieve this dosage will depend upon the severity of the disease andthe general state of the patient's own immune system, but generallyrange from 0.005 to 5.0 mg of selected peptide ligand per kilogram ofbody weight, with doses of 0.05 to 2.0 mg/kg/dose being more commonlyused. For prophylactic applications, compositions containing the presentpeptide ligands or cocktails thereof may also be administered in similaror slightly lower dosages.

A composition containing a peptide ligand according to the presentinvention may be utilised in prophylactic and therapeutic settings toaid in the alteration, inactivation, killing or removal of a selecttarget cell population in a mammal. In addition, the peptide ligandsdescribed herein may be used extracorporeally or in vitro selectively tokill, deplete or otherwise effectively remove a target cell populationfrom a heterogeneous collection of cells. Blood from a mammal may becombined extracorporeally with the selected peptide ligands whereby theundesired cells are killed or otherwise removed from the blood forreturn to the mammal in accordance with standard techniques.

Therapeutic Uses

According to a further aspect of the invention, there is provided aheterotandem bicyclic peptide complex as defined herein for use inpreventing, suppressing or treating cancer.

Examples of cancers (and their benign counterparts) which may be treated(or inhibited) include, but are not limited to tumors of epithelialorigin (adenomas and carcinomas of various types includingadenocarcinomas, squamous carcinomas, transitional cell carcinomas andother carcinomas) such as carcinomas of the bladder and urinary tract,breast, gastrointestinal tract (including the esophagus, stomach(gastric), small intestine, colon, rectum and anus), liver(hepatocellular carcinoma), gall bladder and biliary system, exocrinepancreas, kidney, lung (for example adenocarcinomas, small cell lungcarcinomas, non-small cell lung carcinomas, bronchioalveolar carcinomasand mesotheliomas), head and neck (for example cancers of the tongue,buccal cavity, larynx, pharynx, nasopharynx, tonsil, salivary glands,nasal cavity and paranasal sinuses), ovary, fallopian tubes, peritoneum,vagina, vulva, penis, cervix, myometrium, endometrium, thyroid (forexample thyroid follicular carcinoma), adrenal, prostate, skin andadnexae (for example melanoma, basal cell carcinoma, squamous cellcarcinoma, keratoacanthoma, dysplastic naevus); haematologicalmalignancies (i.e. leukemias, lymphomas) and premalignant haematologicaldisorders and disorders of borderline malignancy includinghaematological malignancies and related conditions of lymphoid lineage(for example acute lymphocytic leukemia [ALL], chronic lymphocyticleukemia [CLL], B-cell lymphomas such as diffuse large B-cell lymphoma[DLBCL], follicular lymphoma, Burkitt's lymphoma, mantle cell lymphoma,T-cell lymphomas and leukaemias, natural killer [NK] cell lymphomas,Hodgkin's lymphomas, hairy cell leukaemia, monoclonal gammopathy ofuncertain significance, plasmacytoma, multiple myeloma, andpost-transplant lymphoproliferative disorders), and haematologicalmalignancies and related conditions of myeloid lineage (for exampleacute myelogenousleukemia [AML], chronic myelogenousleukemia [CML],chronic myelomonocyticleukemia [CMML], hypereosinophilic syndrome,myeloproliferative disorders such as polycythaemia vera, essentialthrombocythaemia and primary myelofibrosis, myeloproliferative syndrome,myelodysplastic syndrome, and promyelocyticleukemia); tumors ofmesenchymal origin, for example sarcomas of soft tissue, bone orcartilage such as osteosarcomas, fibrosarcomas, chondrosarcomas,rhabdomyosarcomas, leiomyosarcomas, liposarcomas, angiosarcomas,Kaposi's sarcoma, Ewing's sarcoma, synovial sarcomas, epithelioidsarcomas, gastrointestinal stromal tumors, benign and malignanthistiocytomas, and dermatofibrosarcomaprotuberans; tumors of the centralor peripheral nervous system (for example astrocytomas, gliomas andglioblastomas, meningiomas, ependymomas, pineal tumors and schwannomas);endocrine tumors (for example pituitary tumors, adrenal tumors, isletcell tumors, parathyroid tumors, carcinoid tumors and medullarycarcinoma of the thyroid); ocular and adnexal tumors (for exampleretinoblastoma); germ cell and trophoblastic tumors (for exampleteratomas, seminomas, dysgerminomas, hydatidiform moles andchoriocarcinomas); and paediatric and embryonal tumors (for examplemedulloblastoma, neuroblastoma, Wilms tumor, and primitiveneuroectodermal tumors); or syndromes, congenital or otherwise, whichleave the patient susceptible to malignancy (for example XerodermaPigmentosum).

In a further embodiment, the cancer is selected from a hematopoieticmalignancy such as selected from: non-Hodgkin's lymphoma (NHL),Burkitt's lymphoma (BL), multiple myeloma (MM), B chronic lymphocyticleukemia (B-CLL), B and T acute lymphocytic leukemia (ALL), T celllymphoma (TCL), acute myeloid leukemia (AML), hairy cell leukemia (HCL),Hodgkin's Lymphoma (HL), and chronic myeloid leukemia (CML).

References herein to the term “prevention” involves administration ofthe protective composition prior to the induction of the disease.“Suppression” refers to administration of the composition after aninductive event, but prior to the clinical appearance of the disease.“Treatment” involves administration of the protective composition afterdisease symptoms become manifest.

Animal model systems which can be used to screen the effectiveness ofthe peptide ligands in protecting against or treating the disease areavailable. The use of animal model systems is facilitated by the presentinvention, which allows the development of polypeptide ligands which cancross react with human and animal targets, to allow the use of animalmodels.

The invention is further described below with reference to the followingexamples.

EXAMPLES

In general, the heterotandem bicyclic peptide complex of the inventionmay be prepared in accordance with the following general method:

All solvents are degassed and purged with N₂ 3 times. A solution ofBP-23825 (1.0 eq), HATU (1.2 eq) and DIEA (2.0 eq) in DMF is mixed for 5minutes, then Bicycle1 (1.2 eq.) is added. The reaction mixture isstirred at 40° C. for 16 hr. The reaction mixture is then concentratedunder reduced pressure to remove solvent and purified by prep-HPLC togive intermediate 2.

A mixture of intermediate 2 (1.0 eq) and Bicycle2 (2.0 eq) are dissolvedin t-BuOH/H₂O (1:1), and then CuSO₄ (1.0 eq), VcNa (4.0 eq), and THPTA(2.0 eq) are added. Finally, 0.2 M NH₄HCO₃ is added to adjust pH to 8.The reaction mixture is stirred at 40° C. for 16 hr under N₂ atmosphere.The reaction mixture was directly purified by prep-HPLC.

More detailed experimental for the heterotandem bicyclic peptide complexof the invention is provided herein below:

Example 1: Synthesis of BCY11863

Procedure for Preparation of BCY12476

A mixture of N-(acid-PEG3)-N-bis(PEG3-azide) (70.0 mg, 112.2 μmol, 1.0eq), HATU (51.2 mg, 134.7 μmol, 1.2 eq) and DIEA (29.0 mg, 224.4 μmol,40 μL, 2.0 eq) was dissolved in DMF (2 mL), and mixed for 5 min. ThenBCY8116 (294.0 mg, 135.3 μmol, 1.2 eq) was added. The reaction mixturewas stirred at 40° C. for 16 hr. LC-MS showed one main peak with desiredm/z. The reaction mixture was concentrated under reduced pressure toremove solvent and produced a residue. The residue was then purified bypreparative HPLC. BCY12476 (194.5 mg, 66.02 μmol, 29% yield, 94% purity)was obtained as a white solid. Calculated MW: 2778.17, observed m/z:1389.3 ([M+2H]²⁺), 926.7 ([M+3H]³⁺).

Procedure for Preparation of BCY11863

A mixture of BCY12476 (100.0 mg, 36.0 μmol, 1.0 eq), BCY8928 (160.0 mg,72.0 μmol, 2.0 eq) were first dissolved in 2 mL of t-BuOH/H₂O (1:1), andthen CuSO₄ (0.4 M, 180 μL, 1.0 eq) and VcNa (28.5 mg, 143.8 μmol, 4.0eq), THPTA (31.2 mg, 71.8 μmol, 2.0 eq) were added. Finally, 0.2 MNH₄HCO₃ was added to adjust pH to 8. All solvents here were degassed andpurged with N₂. The reaction mixture was stirred at 40° C. for 16 hrunder N₂ atmosphere. LC-MS showed BCY8928 remained and desired m/z wasalso detected. The reaction mixture was directly purified by preparativeHPLC. First purification resulted in BCY11863 (117.7 mg, 15.22 μmol,42.29% yield, 93.29% purity) as TFA salt, while less pure fractions werepurified again by preparative HPLC, producing BCY11863 (33.2 mg, 4.3μmol, 12% yield, 95% purity) as TFA salt. Calculated MW: 7213.32,observed m/z: 1444.0 ([M+5H]⁵⁺).

Example 2: Synthesis of BCY13390

Procedure for Preparation of BCY13689

A mixture of BCY12476 (47.0 mg, 16.91 μmol, 1.0 eq), BCY8928 (30.0 mg,13.53 μmol, 0.8 eq), and THPTA (36.7 mg, 84.55 μmol, 5.0 eq) wasdissolved in t-BuOH/H₂O (1:1, 8 mL, pre-degassed and purged with N₂),and then CuSO₄ (0.4 M, 21.0 μL, 0.5 eq) and VcNa (67.0 mg, 338.21 μmol,20.0 eq) were added under N₂. The pH of this solution was adjusted to 8by dropwise addition of 0.2 M NH₄HCO₃ (in 1:1 t-BuOH/H₂O), and thesolution turned light yellow. The reaction mixture was stirred at 25° C.for 1.5 h under N₂ atmosphere. LC-MS showed that some BCY12476 remained,BCY8928 was consumed completely, and a peak with the desired m/z wasdetected. The reaction mixture was filtered and concentrated underreduced pressure to give a residue. The crude product was purified bypreparative HPLC, and BCY13689 (25.3 mg, 4.56 μmol, 27% yield, 90%purity) was obtained as a white solid. Calculated MW: 4995.74, observedm/z: 1249.4 ([M+4H]⁴), 999.9 ([M+5H]*).

Procedure for Preparation of BCY13390

A mixture of BCY13689 (43.6 mg, 8.73 μmol, 1.0 eq), BCY13389 (20.8 mg,9.16 μmol, 1.05 eq), and THPTA (3.8 mg, 8.73 μmol, 1.0 eq) was dissolvedin t-BuOH/H₂O (1:1, 1 mL, pre-degassed and purged with N₂), and thenCuSO₄ (0.4 M, 22.0 μL, 1.0 eq) and VcNa (3.5 mg, 17.45 μmol, 2.0 eq)were added under N₂. The pH of this solution was adjusted to 8 bydropwise addition of 0.2 M NH₄HCO₃ (in 1:1 t-BuOH/H₂O), and the solutionturned to light yellow. The reaction mixture was stirred at 25° C. for 2hr under N₂ atmosphere. LC-MS showed a significant peak corresponding tothe desired m/z. The reaction mixture was filtered and concentratedunder reduced pressure to give a residue. The crude product was purifiedby preparative HPLC, and BCY13390 (33.8 mg, 4.21 μmol, 48% yield, 90%purity) was obtained as a white solid. Calculated MW: 7270.41, observedm/z: 1454.9 ([M+5H]⁵⁺), 1213.2 ([M+6H]⁶⁺).

Example 3: Synthesis of BCY13582

Procedure for Preparation of BCY13582

A mixture of BCY13390 (5.0 mg, 0.6 μmol, 1.0 eq), biotin-PEG12-NHS ester(CAS 365441-71-0, 0.7 mg, 0.72 μmol, 1.1 eq) was dissolved in MeCN/H₂O(1:1, 2 mL). The pH of this solution was adjusted to 8 by dropwiseaddition of 1.0 MNaHCO₃. The reaction mixture was stirred at 25° C. for0.5 hr. LC-MS showed BCY13390 was consumed completely, and one main peakwith desired m/z was detected. The reaction mixture was filtered andconcentrated under reduced pressure to give a residue. The crude productwas purified by preparative HPLC, and BCY13582 (2.5 mg, 0.30 μmol, 43%yield, 96% purity) was obtained as a white solid. Calculated MW:8096.43, observed m/z: 1351.1 ([M+6H]⁶⁺), 1158.5 ([M+7H]⁷⁺).

Example 4: Synthesis of BCY13583

Procedure for Preparation of BCY13583

A mixture of BCY13390 (15.0 mg, 2.06 μmol, 1.0 eq) and Alexa Fluor® 488NHS ester (2.5 mg, 4.12 μmol, 2.0 eq) was dissolved in DMF (0.5 mL).DIEA (2.6 mg, 20.63 μmol, 3.6 μL, 10 eq) was then added dropwise. Thereaction mixture was stirred at 25° C. for 1 hr. LC-MS showed BCY13390remained, and one main peak with desired m/z was detected. AdditionalAlexa Fluor® 488 NHS ester (2.0 mg, 3.09 μmol, 1.5 eq) was added to thereaction mixture, and the reaction mixture was stirred at 25° C. for oneadditional hour. HPLC showed BCY13390 was consumed completely. Thereaction mixture was filtered and concentrated under reduced pressure togive a residue. The crude product was purified by preparative HPLC, andBCY13583 (5 mg, 0.61 μmol, 29% yield, 95% purity) was obtained as a redsolid. Calculated MW: 7787.9, observed m/z: 1948.8 ([M+4H+H₂O]⁴⁺),1558.6 ([M+5H+H₂O]⁵⁺), 1299.1 ([M+7H+H₂O]⁷⁺).

Example 5: Synthesis of BCY13628

Procedure for Preparation of BCY13628

A mixture of BCY13390 (5.6 mg, 0.77 μmol, 1.0 eq) and cyanine 5 NHSester (0.5 mg, 0.85 μmol, 1.1 eq) was dissolved in MeCN/H₂O (1:1, 2 mL).The pH of this solution was adjusted to 8 by dropwise addition of 1.0 MNaHCO₃. The reaction mixture was stirred at 25° C. for 0.5 hr. LC-MSshowed BCY13390 was consumed completely and one main peak with desiredm/z was detected. The reaction mixture was filtered and concentratedunder reduced pressure to give a residue. The crude product was purifiedby preparative HPLC, and BCY13628 (2.9 mg, 0.36 μmol, 46% yield, 95%purity) was obtained as a blue solid. Calculated MW: 7736.06, observedm/z: 1289.9 ([M+6H]⁶⁺), 1105.5 ([M+7H]⁷⁺).

Example 6: Synthesis of BCY15155

Procedure for Preparation of BCY15155

A mixture of BCY13689 (25.0 mg, 5.00 μmol, 1.0 eq), BCY14601 (13.0 mg,6.01 μmol, 1.2 eq), and THPTA (2.0 mg, 5.00 μmol, 1.0 eq) was dissolvedin t-BuOH/0.2 M NH₄HCO₃ (1:1, 0.5 mL, pre-degassed and purged with N₂),and then CUSO₄ (0.4 M, 12.5 μL, 1.0 eq) and Vc (3.5 mg, 20.02 μmol, 4.0eq) were added under N₂. The pH of this solution was adjusted to 8, andthe solution turned light yellow. The reaction mixture was stirred at25° C. for 2 hr under N₂ atmosphere. LC-MS showed BCY13689 was consumedcompletely, some BCY14601 remained and one main peak with desired m/zwas detected. The reaction mixture was filtered and concentrated underreduced pressure to give a residue. The crude product was purified bypreparative HPLC, and BCY15155 (19.7 mg, 2.41 μmol, 36% yield, 97%purity) was obtained as a white solid. Calculated MW:7171.3, observedm/z:1434.7 ([M+5H]⁵⁺), 1196.2 ([M+6H]⁶⁺).

Example 7: Synthesis of BCY14602

Procedure for Preparation of BCY14602

A mixture of BCY12476 (100.0 mg, 36.00 μmol, 1.0 eq), BCY14601 (158.0mg, 72.63 μmol, 2.04 eq), and THPTA (15.6 mg, 36.00 μmol, 1.0 eq) wasdissolved in t-BuOH/0.2 M NH₄HCO₃ (1:1, 2 mL, pre-degassed and purgedwith N₂), and then CUSO₄ (0.4 M, 89.0 μL, 1.0 eq) and VcNa (28.5 mg,143.98 μmol, 4.0 eq) were added under N₂. The pH of this solution wasadjusted to 8, and the solution turned light yellow. THPTA and VcNa werereplenished twice, and overall the solution was stirred at 25° C. for 48hr under N₂ atmosphere. LC-MS showed BCY12476 was consumed completely,BCY14601 remained and one main peak with desired m/z was detected. Somebyproduct was also detected. The reaction mixture was filtered andconcentrated under reduced pressure to give a residue. The crude productwas purified by preparative HPLC, and BCY14602 (45.2 mg, 5.51 μmol, 15%yield, 86% purity) was obtained as a white solid. Calculated MW: 7129.2,observed m/z: 1426.6 ([M+5H]⁵*), 1189.1 ([M+6H]⁶*).

Analytical Data

The following heterotandem bicyclic peptide complexes of the inventionwere analysed using mass spectrometry and HPLC. HPLC setup was asfollows:

-   -   Mobile Phase: A: 0.1% TFA in H₂O B: 0.1% TFA in ACN    -   Flow: 1.0 ml/min    -   Column: Gemini-NX C18 5 um 110A 150*4.6 mm    -   Instrument: Agilent 1200 HPLC-BE(1-614)

Gradients used are 30-60% B over 20 minutes and the data was generatedas follows:

HPLC Retention Complex ID Analytical Data-Mass Spectrometry Time (min)BCY11863 MW: 7213.32, observed m/z: 10.649 1444.0 ([M/5+H]+)

Biological Data

1. CD137 Reporter Assay Co-Culture with Tumor Cells

Culture medium, referred to as R1 media, is prepared by adding 1% FBS toRPMI-1640 (component of Promega kit CS196005). Serial dilutions of testarticles in R1 are prepared in a sterile 96 well-plate. Add 25 μL perwell of test articles or R1 (as a background control) to designatedwells in a white cell culture plate. Tumor cells* are harvested andresuspended at a concentration of 400,000 cells/mL in R1 media. Twentyfive (25) μL/well of tumor cells are added to the white cell cultureplate. Jurkat cells (Promega kit CS196005, 0.5 mL) are thawed in thewater bath and then added to 5 ml pre-warmed R1 media. Twenty five (25)μL/well of Jurkat cells are then added to the white cell culture plate.Incubate the cells and test articles for 6h at 37° C., 5% CO₂. At theend of 6h, add 75 μL/well Bio-Glo™ reagent (Promega) and incubate for 10min before reading luminescence in a plate reader (Clariostar, BMG). Thefold change relative to cells alone (Jurkat cells+Cell line used inco-culture) is calculated and plotted in GraphPad Prism as log(agonist)vs response to determine EC₅₀ (nM) and Fold Induction over background(Max).

The tumor cell type used in co-culture is NCI-H292, CT26 #7, MC38 #13,HT1376, NCI-H322 and T47D which has been shown to express Nectin-4.

Data presented in FIG. 1A shows that the Nectin-4/CD137 heterotandem(BCY11863) induces strong CD137 activation in a CD137 reporter assay andthe activation is dependent on the binding of the heterotandem to CD137.BCY11617, a molecule in which CD137 bicyclic peptide is comprised of allD-amino acids which abrogates binding does not induce CD137 agonism.

A summary of the EC₅₀ (nM) induced by heterotandem bicyclic peptidecomplexes BCY11863 and close analogues in a CD137 reporter assay inco-culture with a Nectin-4-expressing tumor cell line is reported inTable 1 below and visualized in FIG. 1B. This data demonstrates thepotential of BCY11863 to induce CD137 agonism in coculture with celllines that have a range of Nectin-4 expression.

TABLE 1 EC50 (nM) of Fold induction over background induced byNectin-4/CD137 heterotandem bicyclic peptide complexes in a C0137reporter assay Tumor cell Cell Line used in Arithmetic mean EC₅₀ ComplexID line Species Coculture (nM) BCY11863 mouse CT26#07 0.14 ± 0.07BCY11863 mouse MC38#13 0.31 ± 0.26 BCY11863 human NCI-H292 0.28 ± 0.20BCY11863 human HT1376 0.52 ± 0.30 BCY11863 human NCI-H322 0.33 ± 0.21BCY11863 human T47D 0.42 ± 0.24 BCY11863 human MDA-MB-468 0.23 ± 0.01BCY13582 human HT1376 0.58 ± 0.27 BCY13582 human MDA-MB-468 0.34 ± 0.02BCY13583 human HT1376  1.7 ± 0.9 BCY13583 human MDA-MB-468 0.84 ± 0.07

2. Human PBMC Co-Culture (Cytokine Release) Assay

Human and mouse tumor cell lines were cultured according to suppliers'recommendations. Frozen PBMCs from healthy human donors were thawed andwashed one time in room temperature PBS, and then resuspended in R10medium. 100 μl of PBMCs (1,000,000 PBMCs/ml) and 100 μl of tumor cells(100,000 tumor cells/ml) (Effector: Target cell ratio (E:T) 10:1) wereplated in each well of a 96 well flat bottom plate for the co-cultureassay. 100 ng/ml of soluble anti-CD3 mAb (clone OKT3) was added to theculture on day 0 to stimulate human PBMCs. Test, control compounds, orvehicle controls were diluted in R10 media and 50 μL was added torespective wells to bring the final volume per well to 250 μL. Plateswere covered with a breathable film and incubated in a humidifiedchamber at 37° C. with 5% CO2 for three days. Supernatants werecollected 48 hours after stimulation, and human IL-2 and IFNγ weredetected by Luminex. Briefly, the standards and samples were added toblack 96 well plate. Microparticle cocktail (provided in Luminex kit,R&D Systems) was added and shaken for 2 hours at room temperature. Theplate was washed 3 times using magnetic holder. Biotin cocktail was thenadded to the plate and shaken for 1 hour at RT. The plate was washed 3times using magnetic holder. Streptavidin cocktail was added to theplate and shaken for 30 minutes at RT. The plates were washed 3 timesusing magnetic holder, resuspended in 100 μL of wash buffer, shaken for2 minutes at RT, and read using the Luminex 2000. Raw data were analyzedusing built-in Luminex software to generate standard curves andinterpolate protein concentrations, all other data analyses and graphingwere performed using Excel and Prism software. Data represents studieswith 3-5 independent donor PBMCs tested in technical triplicates.

Data presented in FIGS. 2A and 2B demonstrate that the Nectin-4/CD137heterotandem (BCY11863) induces robust IL-2 and IFN-γ cytokine secretionin a PBMC-4T1 co-culture assay. BCY11617 is a negative control thatbinds Nectin-4 but does not bind CD137.

A summary of the EC₅₀ (nM) and maximum IFN-γ cytokine secretion (pg/ml)induced by selected Nectin-4/CD137 heterotandem bicyclic peptidecomplexes in Human PBMC co-culture (cytokine release) assay is reportedin Table 2 below and visualized in FIG. 2C. This demonstrates thepotential of BCY11863 to induce cytokine secretion in the presence of anumber of different tumor cell lines expressing Nectin-4.

TABLE 2 EC₅₀ of IFN-γ cytokine secretion induced by selectedNectin-4/CD137 heterotandem bicyclic peptide complexes in Human PBMC-4T1co-culture (cytokine release) assay Cell Line IL-2 (nM) IFNy (nM) No. ofDonors MC38 #13 0.25 ± 0.08 0.17 ± 0.11 4 (mouse) 4T1-D02 0.16 ± 0.220.04 ± 0.04 4 (mouse) HT1376 0.39 ± 0.29 0.23 ± 0.15 5 (human) T-47D0.20 ± 0.07 0.08 ± 0.06 3 (human) H322 (human) 0.84 ± 0.15 0.85 ± 0.66 3BCY11863 4T1-Parental(Nectin4-) No induction up to 100 nM

3. Pharmacokinetics of the Nectin-4/CD137 Heterotandem BCY11863 in SDRats

Male SD Rats were dosed with the Nectin-4/CD137 heterotandem BCY11863formulated in 25 mM Histidine HCl, 10% sucrose pH 7 by IV bolus, IVinfusion (15 minutes) or subcutaneously. Serial bleeding (about 80 μLblood/time point) was performed via submandibular or saphenous vein ateach time point. All blood samples were immediately transferred intoprechilled microcentrifuge tubes containing 2 μL K2-EDTA (0.5M) asanti-coagulant and placed on wet ice. Blood samples were immediatelyprocessed for plasma by centrifugation at approximately 4° C., 3000 g.The precipitant including internal standard was immediately added intothe plasma, mixed well and centrifuged at 12,000 rpm, 4° C. for 10minutes. The supernatant was transferred into pre-labeled polypropylenemicrocentrifuge tubes, and then quick-frozen over dry ice. The sampleswere stored at 70° C. or below as needed until analysis. 7.5 μL of thesupernatant samples were directly injected for LC-MS/MS analysis usingan Orbitrap Q Exactive in positive ion mode to determine theconcentrations of analyte. Plasma concentration versus time data wereanalyzed by non-compartmental approaches using the Phoenix WinNonlin 6.3software program. C0, C1, Vdss, T½, AUC(0-last), AUC(0-inf),MRT(0-last), MRT(0-inf) and graphs of plasma concentration versus timeprofile were reported. The pharmacokinetic parameters from theexperiment are as shown in Table 3:

TABLE 3 Pharmacokinetic Parameters in SD Rats Dose Dosing T½ Vdss ClpCompound (mg/kg) Route (h) (L/kg) (ml/min/kg) % F BCY11863 1.9 IV Bolus4.1 1.6 7.7 — 3.2 IV Inf 3.1 1.3 9.3 — (15 min) 6.3 Sc 2.5 — — 95%

Data in Table 3 above and FIG. 5 shows that BCY11863 is a low clearancemolecule with volume of distribution larger than plasma volume. Inaddition, the bioavailability from SC dosing of BCY11863 is high inrats.

TABLE 4 Pharmacokinetic Parameters of BCY11863 and potential metabolitesin SD Rat PK study following 100 mg/kg dose administered by IVadministration Cmax AUC T1/2 Clp Analytes (ng/mL) (ng · h/mL) (h) Vdss(L/kg) (ml/min/kg) BCY11863 279540 129863 5.4 2.3 13 BCY15155 2854 12963.1 — — BCY14602 — — — — —

Data in Table 4 and FIG. 14 shows that <1% of BCY11863 gets metabolizedto BCY15155 upon IV administration of BCY11863 to SD rats. Nosignificant conversion to BCY14602 is noted during the first 24h of thestudy.

4. Pharmacokinetics of the Nectin-4/CD137 Heterotandem BCY11863 inCynomolgus Monkey

Non-naïve Cynomolgus Monkeys were dosed via intravenous infusion (15 or30 min) into the cephalic vein with 1 mg/kg of the Nectin-4/CD137heterotandem BCY11863 formulated in 25 mM Histidine HCl, 10% sucrose pH7. Serial bleeding (about 1.2 ml blood/time point) was performed from aperipheral vessel from restrained, non-sedated animals at each timepoint into a commercially available tube containing potassium (K2)EDTA*2H₂O (0.85-1.15 mg) on wet ice and processed for plasma. Sampleswere centrifuged (3,000×g for 10 minutes at 2 to 8° C.) immediatelyafter collection. 0.1 mL plasma was transferred into labelledpolypropylene micro-centrifuge tubes. 5-fold of the precipitantincluding internal standard 100 ng/mL Labetalol & 100 ng/mLdexamethasone & 100 ng/mL tolbutamide & 100 ng/mL Verapamil & 100 ng/mLGlyburide & 100 ng/mL Celecoxib in MeOH was immediately added into theplasma, mixed well and centrifuged at 12,000 rpm for 10 minutes at 2 to8° C. Samples of supernatant were transferred into the pre-labeledpolypropylene microcentrifuge tubes, and frozen over dry ice. Thesamples were stored at −60° C. or below until LC-MS/MS analysis. Analiquot of 40 μL calibration standard, quality control, single blank anddouble blank samples were added to the 1.5 mL tube. Each sample (exceptthe double blank) was quenched with 200 μL IS1 respectively (doubleblank sample was quenched with 200 μL MeOH with 0.5% tritonX-100), andthen the mixture was vortex-mixed well (at least 15 s) with vortexer andcentrifuged for 15 min at 12000 g, 4° C. A 10 μL supernatant wasinjected for LC-MS/MS analysis using an Orbitrap Q Exactive in positiveion mode to determine the concentrations of analyte. Plasmaconcentration versus time data were analyzed by non-compartmentalapproaches using the Phoenix WinNonlin 6.3 software program. CO, Cl,Vdss, T %, AUC(0-last), AUC(0-inf), MRT(0-last), MRT(0-inf) and graphsof plasma concentration versus time profile were reported. Thepharmacokinetic parameters for two bispecific compounds are as shown inTable 5.

TABLE 5 Pharmacokinetic Parameters in cynomolgous monkey Dose T_(1/2)Clp Vdss Compound (mg/kg) Route (h) (ml/min/kg) (L/kg) BCY11863 0.93 IVinfusion 5.3 3.3 0.62 (30 min) 0.97 IV infusion 4.5 4.8 0.91 (15 min)9.4 IV infusion 8.9 3.9 1.1 (15 min)

FIG. 3 shows the plasma concentration vs time curve of BCY11863 from a 2mg/kg IV dose in SD Rat (n=3) and 1 mg/kg IV infusion in cynomolgusmonkey (n=2). BCY11863 has a volume of distribution at steady state(Vdss) of 1.6 L/kg and a clearance of 7.7 mL/min/kg in rats whichresults in a terminal half life of 4.1h. BCY11863 has a volume ofdistribution at steady state (Vdss) of 0.62 L/kg and a clearance of 3.3mL/min/kg in cyno which results in a terminal half life of 5.3 h.Subsequent studies are consistent with these results. The PK parametersfrom the IV study in cyno indicates that this is a low plasma clearancemolecule with volume of distribution similar to total body water.

5. Pharmacokinetics of the Nectin-4/CD137 Heterotandem BCY11863 in CD1Mice

6 Male CD-1 mice were dosed with 15 mg/kg of the Nectin-4/CD137heterotandem BCY11863 formulated in 25 mM Histidine HCl, 10% sucrose pH7 via intraperitoneal or intravenous administration. Serial bleeding(about 80 μL blood/time point) was performed via submandibular orsaphenous vein at each time point. All blood samples were immediatelytransferred into prechilled microcentrifuge tubes containing 2 μLK2-EDTA (0.5M) as anti-coagulant and placed on wet ice. Blood sampleswere immediately processed for plasma by centrifugation at approximately4° C., 3000 g. The precipitant including internal standard wasimmediately added into the plasma, mixed well and centrifuged at 12,000rpm, 4° C. for 10 minutes. The supernatant was transferred intopre-labeled polypropylene microcentrifuge tubes, and then quick-frozenover dry ice. The samples were stored at 70° C. or below as needed untilanalysis. 7.5 μL of the supernatant samples were directly injected forLC-MS/MS analysis using an Orbitrap Q Exactive in positive ion mode todetermine the concentrations of analyte. Plasma concentration versustime data were analyzed by non-compartmental approaches using thePhoenix WinNonlin 6.3 software program. CO, Cl, Vdss, T½, AUC(0-last),AUC(0-inf), MRT(0-last), MRT(0-inf) and graphs of plasma concentrationversus time profile were reported.

FIG. 9 shows the plasma concentration vs time curves of BCY11863 from a15 mg/kg IP dose in CD1 mice (n=3) and the terminal plasma half life forBCY11863.

TABLE 6 Pharmacokinetic Parameters in CD-1 Mice Dose Dosing T½ Vdss ClpCompound (mg/kg) Route (h) (L/kg) (ml/min/kg) % F BCY11863 5.6 IV Bolus2.6 1.6 9.7 0.96 IV Bolus 1.7 2.9 21 12 IV Bolus 2.6 2.5 17 32 IV Bolus2.4 2.1 16 15.5 IP 2.5 — — 100

Data in FIG. 9 and Table 6 above shows BCY11863 can be dosed as IV bolusand IP in mice. The bioavailability from IP dosing of BCY11863 is highin mice. The PK parameters from the IV study indicates that this is alow clearance molecule with volume of distribution larger than plasmavolume.

6. Anti-Tumor Activity of BCY11863 in a Syngeneic Nectin-4Overexpressing MC38 Tumor Model (MC38#13)

6-8 weeks old C57BL/6J-hCD137 female mice were inoculated in the flankwith 1×10⁶ syngeneic Nectin-4 overexpressing MC38 cells (MC38#13). Whentumors reached 72 mm³ size on average, mice were randomized to receivevehicle or BCY11863 (intraperitoneal administration). BCY11863 wasadministered (n=6 mice/treatment cohort) at either 1 mg/kg or 10 mg/kgeither daily (QD) or every three days (Q3D). QD dosed mice received 16doses of BCY11863 and Q3D dosed mice received 10 doses of BCY11863.Tumor growth was monitored by caliper measurements until day 69 aftertreatment initiation. The results of this experiment may be seen in FIG.4 where significant reduction (p<0.05, 2-way ANOVA with Dunnett's testfor multiple comparisons) of tumor growth was observed in 2 treatmentcohorts by day 7 and by day 14 all treatment groups were significantlydifferent from the vehicle group. By day 48, 22 out of 24BCY11863-treated animals had responded to the treatment completely andhad no palpable tumors remaining.

Based on the circulating plasma half-life of BCY11863 in mice after IPinjection (2.5 h), plasma trough levels will be close to 0 after bothBCY11863 doses (1 and 10 mg/kg) and dosing intervals (QD and Q3D) thusdemonstrating that less than continuous plasma exposure of BCY11863 fromintermittent dosing is sufficient to lead to significant anti-tumoractivity leading to durable complete responses.

7. BCY11863 Treatment Leads to an Immunogenic Memory to Nectin-4Overexpressing MC38 Tumor Model

On day 69, 5 animals that had responded completely to BCY11863 treatmentwere re-inoculated with 1×10⁶ MC38#13-cells. A cohort of 5 naïveC57BL/6J-hCD137 female mice were inoculated with 1×10⁶ MC38#13-cells asa control. The results of this experiment may be seen in FIG. 5 whereall 5 inoculated naïve C57BL/6J-hCD137 female mice grew tumors by day 13after inoculation whereas none of the inoculated complete responder micedeveloped tumors. This demonstrates that animals that achieved acomplete antitumor response as a result of BCY11863 treatment havedeveloped immunogenic memory.

8. BCY11863 Demonstrates Anti-Tumor Activity in a Syngeneic Nectin-4Overexpressing CT26 Tumor Model (CT26#7)

6-8 weeks old BALB/c-hCD137 female mice were inoculated in the flankwith 3×10⁵ syngeneic Nectin-4 overexpressing CT26 cells (CT26#7). Whentumors reached around 70 mm³ size on average, mice were randomized toreceive vehicle or 5 mg/kg BCY11863 intraperitoneally every three days(6 doses total). Tumor growth was monitored by caliper measurementsuntil day 14 after treatment initiation. The results of this experimentmay be seen in FIG. 6 where BCY11863 treatment significantly (p<0.0001,Student's t-test) reduced the tumor growth from day 7 forward.

Based on the circulating plasma half-life of BCY11863 in mice at IPinjection (2.5 h), plasma exposure will not be continuous throughout thedosing period demonstrating that less than continuous plasma exposure ofBCY11863 is sufficient to lead to significant anti-tumor activity.

9. Total T Cells and CD8+ T Cells Increase in CT26#7 Tumor Tissue 1hafter the Last (6^(th)) Q3D Dose of BCY11863

1 hour after the last vehicle or BCY11863 dose the CD26#7 bearing micewere sacrificed and tumors were harvested, processed for single cellsuspensions and stained for flow cytometry analysis for total T cells(CD45+CD3+), CD8+ T cells (CD45+CD3+CD8+), CD4+ T cells (CD45+CD3+CD4+)and regulatory T cells (Tregs; CD45+CD3+CD4+Foxp3+). The results of thisexperiment may be seen in FIG. 7 where it can be seen that BCY11863treatment led to significant increase of total T cells (p<0.0001,Student's t-test) and CD8+ T cells (p<0.0001, Student's t-test) as wellas to a significant increase in the CD8+ T cell/Treg ratio (p<0.05,Student's t-test).

This demonstrates that treatment with BCY11863 can lead to an increasedlevel of T-cells locally in the tumor tissue after intermittent dosing.

10. Pharmacokinetic Profiles of BCY11863 in Plasma and Tumor Tissue ofCT26#7 Syngeneic Tumor Bearing Animals after a Single Intravenous (iv)Administration of 5 mg/kg of BCY11863

6-8 weeks old BALB/c female mice were inoculated in the flank with 3×10⁵syngeneic Nectin-4 overexpressing CT26 cells (CT26#7). When tumorsreached around 400 mm³ size on average, mice were randomized to receivea single intravenous dose of vehicle or 5 mg/kg BCY11863. A cohort ofmice (n=3/timepoint) were sacrificed at 0.25, 0.5, 1, 2, 4, 8 and 24htimepoints and harvested plasma and tumor tissue were analyzed forBCY11863. For tumor BCY11863 content analysis, tumor homogenate wasprepared by homogenizing tumor tissue with 10 volumes (w:v) ofhomogenizing solution (MeOH/15 mM PBS (1:2, v:v)). 40 μL of sample wasquenched with 200 μL IS1 and the mixture was mixed by vortexing for 10min at 800 rpm and centrifuged for 15 min at 3220 g at 4° C. Thesupernatant was transfer to another clean 96-well plate and centrifugedfor 5 min at 3220 g at 4° C., and 10.0 μL of supernatant was theninjected for LC-MS/MS analysis using an Orbitrap Q Exactive in positiveion mode to determine the concentrations of analyte. For plasma BCY11863content analysis, blood samples were collected in K2-EDTA tubes andimmediately processed to plasma by centrifugation at approximately 4°C., 3000 g. 40 μL of plasma sample was quenched with 200 μL IS1 and themixture was mixed by vortexing for 10 min at 800 rpm and centrifuged for15 min at 3220 g at 4° C. The supernatant was transferred to anotherclean 96-well plate and centrifuged for 5 min at 3220 g at 4° C., and10.0 μL of supernatant was then injected for LC-MS/MS analysis using anOrbitrap Q Exactive in positive ion mode to determine the concentrationsof analyte.

The results of this experiment are shown in FIG. 8 where it can be seenthat BCY11863 was retained in the tumor tissue after the plasma BCY11863is eliminated from circulation as indicated by the difference ofBCY11863 plasma T_(1/2) (1.65h) and tumor T_(1/2) (13.4h).

11. Binding of BCY11863 to Nectin-4 and CD137 Across Four PreclinicalSpecies

The binding of BCY11863 to its primary target Nectin-4 and CD137 wascharacterized using surface plasmon resonance (SPR).

(a) Nectin-4

BCY11863 binds to cyno, rat, mouse and human Nectin-4 with K_(D) between5-27 nM as measured by direct binding to the extracellular domain thathas been biotinylated and captured on a streptavidin sensor chipsurface.

TABLE 7 Binding affinities of BCY11863 to Biotinylated-Nectin-4extracellular domain: SPR data SPR K_(D) Assay Human Human NHP Rat Mouse(nM) Type (25° C.) (37° C.) (25° C.) (25° C.) (25° C.) BCY11863 Direct5.0 ± 2.1 5.2 ± 1.1 27 ±15 15 ± 1 4.6 ± 2.1 Binding n = 7 n = 9 n = 9 n= 6 n = 9

To understand whether the binding of BCY11863 to Nectin-4 was altered inthe context of the ternary complex, i.e. when also bound to CD137, amulticomponent SPR binding assay was developed. BCY11863 was firstcaptured to human CD137 immobilized on the SPR chip surface and thenNectin-4 from different species were passed over the chip to determinetheir affinities to the captured BCY11863 (see FIG. 10C). The affinitiesto Nectin-4 were generally maintained in the presence of CD137 bindingas shown below:

TABLE 8 Binding affinities of BCY11863 to Nectin-4 extracellular domainusing biotinylated human CD137 as capture reagent Assay Mouse SPR K_(D)(nM) Type Human NHP Rat 10 BCY11863 Sandwich 12 ± 2 28 ± 5 25 ± 2 6.7 ±1.7 Assay n = 4 n = 3 n = 3 n = 3

(b) CD137

Direct binding of BCY11863 to surface bound CD137 cannot be measuredaccurately by SPR because of avidity resulting from two CD137 bindingbicycles in BCY11863 which leads to extremely slow k_(off) (See FIG.10B). In addition, biotinylation of cyno CD137 abrogates binding ofBCY11863, likely due to modification of a lysine on the cyno proteinthat is important for BCY11863 binding. Hence, a BCY11863 analoguecontaining a C-terminal biotinylated lysine (BCY13582) was tested in SPRto determine cross species specificity of BCY11863. BCY13582 wascaptured to the sensor chip using a reversible biotin capture kit andthe affinities to Nectin-4 from different species were determined. Bothstrategies showed that these BCY11863 analogs bound to human and cynoCD137 with K_(D)<10 nM and had negligible binding to both mouse and ratCD137.

TABLE 9 Binding affinities of biotinylated BCY11863 analogues to CD137extracellular domain: SPR data SPR K_(D) Assay (nM) Type Human NHP RatMouse BCY13582 Direct 8.4 ± 4.2 4.23 NB NB Binding n = 3 n = 1 n = 1 n =1

To understand whether the binding of BCY11863 to CD137 was altered inthe context of the ternary complex, i.e. when also bound to Nectin-4, adual binding SPR binding assay was developed. BCY11863 was firstcaptured to human Nectin-4 immobilized on the SPR chip surface and thensoluble CD137 from different species were passed over the chip todetermine their affinities to the captured BCY11863 (see FIG. 10D). Theaffinities to CD137 were generally maintained in the presence ofNectin-4 binding as shown below:

TABLE 10 Binding affinities of BCY11863 to CD137 ECD using biotinylatedhuman Nectin-4 as capture reagent SPR K_(D) Assay (nM) Type Human NHPRat Mouse BCY11863 Dual 6.3 ± 0.7 18 ± 6 NB NB Binding n = 4 n = 3 n = 2n = 2

FIG. 10A shows one example sensorgram which demonstrates that BCY11863binds to Nectin-4 (human) with an affinity of 4.1 nM. FIG. 10B shows thesensorgram that BCY11863 binds to CD137 (human) with high affinity. Dueto the presence of 2 CD137 binding bicycles in BCY11863, the off ratefrom immobilized CD137 protein is very slow and the reported K_(D) maybe an overestimation (FIG. 10B). FIG. 10C shows BCY11863 binds toNectin-4 while the CD137 arms are bound to CD137 protein immobilized onthe chip to form a ternary complex. FIG. 10D shows BCY11863 binds toCD137 while the Nectin-4 binding arm is bound to Nectin-4 proteinimmobilized on the chip to form a ternary complex. FIG. 10E demonstratesthe ability of BCY13582 immobilized on SPR chip to bind human CD137.

12. Selectivity of BCY11863 for Nectin-4 and CD137

Nectin—4 Paralogue screening: Binding of BCY11863 was assessed using SPRagainst Nectin-1 (2880-N1, R&D Systems), Nectin-2 (2229-N2, R&DSystems), Nectin-3 (3064-N3, R&D Systems), Nectin-like-1 (3678-S4-050,R&D Systems), Nectin-like-2 (3519-S4-050, R&D Systems), Nectin-like-3(4290-S4-050, R&D Systems), Nectin-like-4 (4164-S4, R&D Systems) andNectin-like-5 (2530-CD-050, R&D Systems) by labelling them with biotinand immobilizing them on a streptavidin surface. BCY11863 did not showany binding to these targets up to a concentration of 5000 nM.

CD137 Paralogue screening: Binding of streptavidin captured BCY13582(biotinylated-BCY11863) was assessed using SPR against soluble TNFfamily receptors OX40 and CD40. BCY13582 did not bind to these targetsup to a concentration of 100 nM.

Retrogenix microarray screening: Retrogenix's cell microarray technologywas used to screen for specific off-target binding interactions of abiotinylated BCY11863 known as BCY13582.

Investigation of the levels of binding of the test peptide to fixed,untransfected HEK293 cells, and to cells over-expressing Nectin-4 andCD137 (TNFRSF9), showed 1 μM of the test peptide to be a suitablescreening concentration. Under these conditions, the test peptide wasscreened for binding against human HEK293 cells, individually expressing5484 full-length human plasma membrane proteins and secreted proteins.This revealed 9 primary hits, including Nectin-4 and CD137.

Each primary hit was re-expressed, along with two control receptors(TGFBR2 and EGFR), and re-tested with 1 μM BCY13582 test peptide, 1 μMBCY13582 test peptide in the presence of 100 μM BCY11863, and otherpositive and negative control treatments (FIG. 4). After removingnon-specific, non-reproducible and non-significant hits, there remainedthree specific interactions for the test peptide. These were untetheredand tethered forms of Nectin-4, and CD137—the primary targets.

No specific off-target interactions were identified for BCY13582,indicating high specificity for its primary targets.

13. Anti-Tumor Activity of BCY11863 in a Syngeneic Nectin-4Overexpressinq MC38 Tumor Model (MC38#13) on Dosing on Twice a Week at 5mg/kg at 0, 24h and 10 mg/kg at 0h

6-8 week old female C57BL/6J-hCD137 mice [B-hTNFRSF9(CD137) mice;Biocytogen] were implanted subcutaneously with 1×10⁶ MC38#13 (MC38 cellsengineered to overexpress murine Nectin-4) cells. Mice were randomizedinto treatment groups (n=6/cohort) when average tumor volumes reachedaround 95 mm³ and were treated with a weekly dose of vehicle (25 mMhistidine, 10% sucrose, pH7) or 10 mg/kg BCY11863 with two differentdosing schedules for two dosing cycles (5 mg/kg BCY11863 at 0h and 24hon DO and D7, or 10 mg/kg at 0h on DO and D7). All treatments wereadministered intravenously (IV). Tumor growth was monitored until Day 15from treatment initiation.

BCY11863 leads to significant anti-tumor activity with both dosingschedules, but the dose schedule with 5 mg/kg dosing at 0h and 24h wassuperior to 10 mg/kg dosing at 0h when complete responses were analyzedon day 15 after treatment initiation (FIG. 12). 5 mg/kg BCY11863 at 0hand 24h on DO and D7 dosing led to 4 out of 6 complete tumor responseswhereas 10 mg/kg BCY11863 at 0h on DO and D7 dosing led to one out of 6complete tumor responses. These data together with the BCY11863 mouseplasma PK data indicate that maintaining a BCY11863 plasma exposure atthe level produced by 5 mg/kg 0h and 24h dosing in a weekly cycleproduces close to complete anti-tumor response in the MC38#13 tumormodel.

14. Anti-Tumor Activity of BCY11863 in a Syngeneic Nectin-4Overexpressing MC38 Tumor Model (MC38#13)

At 3 weekly doses of 3, 10 and 30 mg/kg with dose fractionated weekly,biweekly and daily 6-8 week old female C57BL/6J-hCD137 mice[B-hTNFRSF9(CD137) mice; Biocytogen] were implanted subcutaneously with1×10⁶ MC38#13 (MC38 cells engineered to overexpress murine Nectin-4)cells. Mice were randomized into treatment groups (n=6/cohort) whenaverage tumor volumes reached around 107 mm³ and were treated with 21daily doses of vehicle (25 mM histidine, 10% sucrose, pH7). BCY11863treatment was done at three different total dose levels (3, 10 and 30mg/kg total weekly dose) fractionated in three different schedules (QD:daily; BIW: twice a week or QW: weekly). Different BCY11863 treatmentcohorts received either 21 daily doses (0.43, 1.4 or 4.3 mg/kg), 6 twiceweekly doses (1.5, 5 or 15 mg/kg) or 3 weekly doses (3, 10 or 30 mg/kg).All treatments were administered intravenously (IV). Tumor growth wasmonitored until tumor reached volumes over 2000 mm³ or until 31 daysafter treatment initiation. Complete responders (animals with nopalpable tumors) were followed until D52.

BCY11863 leads to significant anti-tumor activity with many of thedosing schedules the BIW dosing schedule being the most efficaciousschedule, the 5 mg/kg BIW dose in particular. This is demonstrated bythe number of complete responder animals on day 52. On day 52 aftertreatment initiation, 15/18 mice treated BIW with BCY11863 were completeresponders, 12/18 mice treated QD with BCY11863 were complete respondersand 6/18 mice treated QW with BCY11863 were complete responders. 5 mg/kgBIW dosing lead to 100% complete response rate with 6/6 CRs (FIG. 13).These data together with the BCY11863 mouse plasma PK data indicate thatcontinuous BCY11863 plasma exposure is not needed for anti-tumorresponse to BCY11863 in the MC38#13 tumor model.

1. A heterotandem bicyclic peptide complex comprising: (a) a firstpeptide ligand which binds to Nectin-4 and which has the sequenceC_(i)P[1Nal][dD]C_(ii)M[HArg]DWSTP[HyP]WC_(iii) (SEQ ID NO: 1);conjugated via an N-(acid-PEG₃)-N-bis(PEG₃-azide) linker to (b) twosecond peptide ligands which bind to CD137 both of which have thesequence Ac—C_(i)[tBuAla]PE[D-Lys(PYA)]PYC_(ii)FADPY[Nle]C_(iii)-A (SEQID NO: 2); or a pharmaceutically acceptable salt thereof, wherein eachof said peptide ligands comprise a polypeptide comprising three reactivecysteine groups (C_(i), C_(ii) and C_(iii)), separated by two loopsequences, and a molecular scaffold which is1,1′,1″-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA) andwhich forms covalent bonds with the reactive cysteine groups of thepolypeptide such that two polypeptide loops are formed on the molecularscaffold; wherein Ac represents acetyl, HArg represents homoarginine,HyP represents trans-4-hydroxy-L-proline, 1Nal represents1-naphthylalanine, tBuAla represents t-butyl-alanine, PYA represents4-pentynoic acid and Nle represents norleucine.
 2. The heterotandembicyclic peptide complex according to claim 1 which is:

or a pharmaceutically acceptable salt thereof.
 3. The heterotandembicyclic peptide complex as defined in claim 1, wherein thepharmaceutically acceptable salt is selected from the free acid or thesodium, potassium, calcium, ammonium salt.
 4. A pharmaceuticalcomposition which comprises the heterotandem bicyclic peptide complex ofclaim 1, or a pharmaceutically acceptable salt thereof, in combinationwith one or more pharmaceutically acceptable excipients.
 5. A method ofpreventing, suppressing or treating cancer, comprising administering theheterotandem bicyclic peptide complex as defined in claim 1, or apharmaceutically acceptable salt thereof.
 6. A method of treating cancerwhich comprises administration of the heterotandem bicyclic peptidecomplex as defined in claim 1, or a pharmaceutically acceptable saltthereof, at a dosage frequency which does not sustain plasmaconcentrations of said complex above the in vitro EC₅₀ of said complex.7. The method of claim 5, wherein the cancer is lung cancer, coloncancer, bladder cancer, or breast cancer.
 8. The method of claim 6,wherein the cancer is lung cancer, colon cancer, bladder cancer, orbreast cancer.
 9. The method of claim 6, wherein the dosage frequency isonce per week.
 10. The method of claim 6, wherein the dosage frequencyis twice per week.
 11. A heterotandem bicyclic peptide complex which is:

or a pharmaceutically acceptable salt thereof.
 12. The heterotandembicyclic peptide complex of claim 11, wherein the pharmaceuticallyacceptable salt is selected from the free acid or the sodium, potassium,calcium, ammonium salt.
 13. A pharmaceutical composition comprising theheterotandem bicyclic peptide complex of claim 11, or a pharmaceuticallyacceptable salt thereof, in combination with one or morepharmaceutically acceptable excipients.
 14. A method of preventing,suppressing or treating cancer, comprising administering theheterotandem bicyclic peptide complex of claim 11, or a pharmaceuticallyacceptable salt thereof.
 15. The method of claim 14, wherein the canceris lung cancer, colon cancer, bladder cancer, or breast cancer.
 16. Amethod of treating cancer which comprises administration of theheterotandem bicyclic peptide complex of claim 11, or a pharmaceuticallyacceptable salt thereof, at a dosage frequency which does not sustainplasma concentrations of said complex above the in vitro EC₅₀ of saidcomplex.
 17. The method of claim 16, wherein the cancer is lung cancer,colon cancer, bladder cancer, or breast cancer.
 18. The method of claim16, wherein the dosage frequency is once per week.
 19. The method ofclaim 16, wherein the dosage frequency is twice per week.